The insights into the evolutionary history of Translucidithyrium: based on a newly-discovered species

Abstract During the field studies, a Translucidithyrium-like taxon was collected in Xishuangbanna of Yunnan Province, during an investigation into the diversity of microfungi in the southwest of China. Morphological observations and phylogenetic analysis of combined LSU and ITS sequences revealed that the new taxon is a member of the genus Translucidithyrium and it is distinct from other species. Therefore, Translucidithyriumchinensesp. nov. is introduced here. The Maximum Clade Credibility (MCC) tree from LSU rDNA of Translucidithyrium and related species indicated the divergence time of existing and new species of Translucidithyrium was crown age at 16 (4–33) Mya. Combining the estimated divergence time, paleoecology and plate tectonic movements with the corresponding geological time scale, we proposed a hypothesis that the speciation (estimated divergence time) of T.chinense was earlier than T.thailandicum. Our findings provided new insights into the species of Translucidithyrium about ecological adaptation and speciation in two separate areas.


Introduction
The sooty blotch and flyspeck fungi are widespread species and commonly occur on the surface of leaves, stems and fruits in tropical and subtropical zones (Yang et al. 2010;Gleason et al. 2011;Hongsanan et al. 2017;Zeng et al. 2018). Although these fungi do not directly harm host plants, they may affect the economic value of fruit sales ability and reduce photosynthesis in plants (Gleason et al. 2011). Sooty blotch fungi can form dark mycelial mats, whereas flyspeck fungi lack mycelial mats, form shiny and small, black spots (Batzer et al. 2005;Yang et al. 2010;Gleason et al. 2011;Zhang et al. 2015;Singtripop et al. 2016;Hongsanan et al. 2017). However, these fungi are poorly known, because of the difficulty in obtaining the strain which grows slowly (Yang et al. 2010;Hongsanan et al. 2017;Zeng et al. 2018).
Translucidithyrium X.Y. Zeng & K.D. Hyde (2018) was introduced as a monotypic genus in Phaeothecoidiellaceae, which is represented by T. thailandicum X.Y. Zeng & K.D. Hyde (2018). It was characterised by epiphytes on the reverse of living leaves, semi-transparent ascomata, globose to subglobose asci and fusiform ascospores with verrucose and appendages. Ascospores germinated on MEA (Malt Extract Agar Medium) within 24 h. The colonies slowly grow on media, white to grey, circular and villiform (Zeng et al. 2018). Liu et al. (2017) used the molecular clock approach to estimate the divergence time of the order Capnodiales crown age at 151-283 Mya (million years ago). Zeng et al. (2019) estimated the divergence time of the family Phaeothecoidiellaceae crown age at 40-60 Mya. The molecular clock approach for estimating divergence time might be used to predict speciation, historical climate change or other environmental events (Hélène and Arne 2014;Louca and Pennell 2020).
In this study, we collected an extraordinary new species of Translucidithyrium in Xishuangbanna, Yunnan Province, China. We described the morphological characteristics and built a phylogenetic tree to determine the classification of the new taxon. We compared and analysed the estimated divergence time of Translucidithyrium with the environmental changes around the corresponding time range to propose the evolutional history hypothesis of Translucidithyrium distributed in two different regions (China and Thailand).

Morphological
Fresh living leaves with olivaceous dots were collected at Xishuangbanna, China 21°55'51"N, 101°15'08"E, 540 m alt.) and delivered to the laboratory for observation. According to Wu et al. (2014), the collected samples were processed and examined by microscopes: the photos of ascomata were taken by using a compound stereomicroscope (KEYENCE CORPORATION V.1.10 with camera VH-Z20R). Hand sections were made under a stereomicroscope (OLYMPUS SZ61) and mounted in water and blue cotton and photomicrographs of fungal structures were taken with a compound microscope (Nikon ECLIPSE 80i). The single spore isolation was implemented by the methods of Choi et al. (1999) and Chomnunti et al. (2014). Germinated spores were individually transferred to PDA (Potato Dextrose Agar Medium) and incubated at 26 °C for 48 h. Colony characteristics were observed and measured after 4 weeks at 26 °C. Images used for figures were processed with Adobe Photoshop CC v. 2015.5.0 software (Adobe Systems, USA). The holotype was deposited at the herbarium of IFRD (International Fungal Research & Development Centre; Research Institute of Resource Insects, Kunming), reference number IFRD 9208. The ex-type strain was deposited at IFRDCC, reference number IFRDCC 3000.

DNA isolation, amplification and sequencing
According to the manufacturer's instructions, genomic DNA was extracted from mycelium growing on PDA at room temperature by using the Forensic DNA Kit (OMEGA, USA). The primer pair LR0R and LR5 was used to amplify the large subunit (LSU) rDNA (Vilgalys and Hester 1990). The primer pair ITS5 and ITS4 was used to amplify the internal transcribed spacer (ITS) rDNA (White et al. 1990). The primer pair NS1 and NS4 was used to amplify the partial small subunit (SSU) rDNA (White et al. 1990). The PCR reactions were in accordance with instructions from Golden Mix, Beijing TsingKe Biotech Co. Ltd, Beijing, China: initial denaturation at 98 °C for 2 min, then 30 cycles of 98 °C denaturation for 10 s, 56 °C annealing for 10 s and 72 °C extension for 10 s (ITS and SSU) or 20 s (LSU) and a final extension at 72 °C for 1 min. All PCR products were sequenced by Biomed (Beijing, China).
Maximum Likelihood (ML) analysis was conducted by using RAxMLGUI v.1.0 (Silvestro and Michalak 2012). Aligned sequences were input into the software and Dothidea sambuci was selected as the outgroup taxon. One thousand non-parametric bootstrap iterations were employed with the "ML + rapid bootstrap" tools and "GTR-GAMMA" arithmetic.
For Bayesian analysis, MrModeltest 2.3 (Nylander 2004) was used to estimate the best-fitting model for the combined LSU and ITS genes. Posterior probabilities (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo (MCMC) sampling in MrBayes v.3.2 (Ronquist and Huelsenbeck 2003). Six simultaneous Markov chains were run for 2,000,000 generations; trees were printed every 1,000 generations; trees were sampled every 100 generations. The first 5,000 trees submitted to the burn-in phase and were discarded; the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (Cai et al. 2006(Cai et al. , 2008Liu et al. 2012).

Fossil calibrations and divergence time estimations
The fossil Protographum luttrellii (Renard et al. 2020) was used to calibrate the divergence time of Asterotexiales and Aulographaceae (normal distribution, mean = 119.0, SD = 3.7). The secondary calibration from the family Phaeothecoidiellaceae with a crown age of 58 Mya (normal distribution, mean = 50.0, SD = 6.1) was used (Zeng et al. 2019). The additional secondary calibration of Capnodiales was used, based on the result from Liu et al. (2017) (normal distribution, mean = 217.0, SD = 40.0).
Divergence time analysis was carried out using BEAST v1.8.4 (Drummond et al. 2012). Aligned LSU sequence data were loaded into the BEAUti v1.10.4 for generating an XML file. An uncorrelated relaxed clock model (Drummond et al. 2006) with a lognormal distribution of rates was used for the analysis. We used a Yule Process tree prior (Yule 1925;Gernhard 2008), which assumes a constant speciation rate per lineage and a randomly-generated starting tree. The length of chain was set as 50 million generations and sampling parameters were set at every 5,000 generations in MCMC. Subsequent divergence time analysis was carried out using BEAST v.1.10.4 (Drummond et al. 2012). Tracer v.1.7.1 was used to check the effective sample sizes (ESS) and acceptable values were higher than 200. The .log files and .tree files generated by BEAST were combined in LogCombiner v1.10.4 after removing a proportion of states as burn-in. The Maximum Clade Credibility (MCC) tree was given by obtained data and was estimated in TreeAnnotator v.1.10.4 (Liu et al. 2017;Zeng et al. 2019Zeng et al. , 2020Renard et al. 2020).

Figure 2.
Translucidithyrium chinense (IFRD 9208, holotype) A plant leaves B acscoma on leaves surface C squash of ascoma at 20 times amplification D cross section of ascoma in blue cotton at 20 times amplification E, F cross section of ascoma in blue cotton at 40 times amplification G asci at 100 times amplification H-K asci in blue cotton at 100 times amplification L ascospore at 100 times amplification M-P ascospore in blue cotton at 100 times amplification. Scale bars: 200 μm (B); 100 μm (C, D); 50 μm (E, F); 20 μm (G-K); 10 μm (L-P). We slightly adjusted the contrast, saturation and hue of images and removed the contaminants around main object in images in PS software without obscuration, erasure or distortion of any information existing in the original document. state, lacking pedicel, paraphyses absent ( Fig. 2G-K). Ascospores 41-65 × 10-13 μm (x = 50 × 11 μm, n = 20), irregularly overlapping, hyaline, ovoid at young state, fusiform with both ends tapered at mature state, 1-septate, constricted at the septum, upper cell a little larger than lower, with guttules at both ends, verrucose ( Fig. 2L-P). Asexual morph: Undetermined. Culture characteristics. Ascospores germinating on MEA at 36 h after sporeisolation, germinating on PDA at 48 h after spore-isolation. Colonies slow growing on MEA and PDA, irregular, villiform, convex, white on surface, yellow to brown at base. After a long period of growth, the pigments produced by culture discolour the medium, roots generate at the bottom (Fig. 3A-D). Culture hyphae hyaline, branched, constricted at the septum, 3 μm wide (Fig. 3E, F).
Notes. This new species is morphologically similar to Translucidithyrium thailandicum in having semi-transparent and largish ascomata, globose asci and hyaline ascospores with 1-septate. However, Translucidithyrium chinense has a slightly papilla thyriothecium with weaker transmittance and ascospores with guttules at both ends, while T. thailandicum has a flattened thyriothecium with higher transmittance and ascospores with appendages at both ends; besides, the size of ascomata and asci of T. chinense are slightly larger than those of T. thailandicum (795 μm vs. 621 μm; 77 μm vs. 64 μm). The culture characteristics of both species are different: the culture of T. chinense grows more slowly, has roots inserting into medium and turn the bottom brown. Phylogenetically, T. chinense clusters with T. thailandicum as a distinct clade with high support (100% ML / 1.00 PP, Fig. 1).
Divergence times estimates. The Maximum Clade Credibility (MCC) tree was similar to the major lineages in the Bayesian and ML trees. The crown age of  Translucidithyrium showed 16 Mya (4-33), which was earlier than the divergence time of most genera in Phaeothecoidiellaceae. The estimated divergence time of Phaeothecoidiellaceae from Zeng et al. (2019) is 58 Mya, which corresponds to our results.

Discussion
Translucidithyrium thailandicum was found in the north of Thailand (Zeng et al. 2018). Translucidithyrium chinense was found in the Xishuangbanna Region, southwest of China, which lies on the northern border of a rainforest with rich microfungal resources. The new species is characterised by brown to olivaceous ascomata and slightly semitransparent, subglobose asci without pedicel and fusiform ascospores with verrucose and guttules (Fig. 2). T. chinense is introduced as a new species in Translucidithyrium by morphological and phylogenetic studies (Figs 1-3). The ascomata of Translucidithyrium are different from related genera of Phaeothecoidiellaceae: Nowamyces has immersed ascomata, Chaetothyrina has ascomata with setae and Rivilata has subcuticular ascomata (Singtripop et al. 2016;Doilom et al. 2018;Zeng et al. 2018;Crous et al. 2019;Hongsanan et al. 2020). Translucidithyrium is similar to the family Schizothyriaceae in having semi-transparent ascomata, globose to subglobose asci and hyaline ascospores with guttules. Schizothyriaceae includes Schizothyrium, Plochmopeltis, Hexagonella, Lecideopsella, Mycerema, Kerniomyces, Metathyriella, Myriangiella, Amazonotheca and Vonarxella (Phookamsak et al. 2016;Wijayawardene et al. 2020). The morphology of T. chinense is most similar to Lecideopsella by having globose asci and 1-septate ascospores, but Lecideopsella has a short pedicel at the bottom of the asci (Phookamsak et al. 2016;Zeng et al. 2018). Phylogenetically, Translucidithyrium formed a long clade and clustered within the family Phaeothecoidiellaceae. It indicated the existing certain genetic distance amongst Translucidithyrium, Phaeothecoidiellaceae and Schizothyriaceae. Phaeothecoidiellaceae and Schizothyriaceae are poorly studied families (Batzer et al. 2008;Phookamsak et al. 2016;Singtripop et al. 2016;Hongsanan et al. 2017;Zeng et al. 2018). Therefore, more fresh specimens with molecular data are needed to confirm the classification of Translucidithyrium, Phaeothecoidiellaceae and Schizothyriaceae. Zuckerkandl and Pauling (1962) suggested that the number of differences amongst amino acids was proportional to species divergence time. We estimated the divergence time using BEAST analysis. The divergence time of Translucidithyrium crown age was estimated at 16 Mya (4-33), which was earlier than the crown ages of Chaetothyrina at 2 Mya (0-5), the crown ages of Repetophragma at 9 Mya (2-20), the crown ages of Nowamyces at 7 Mya (1-20) and the crown ages of Phaeothecoidiella at 4 Mya (0-14) within Phaeothecoidiellaceae (Fig. 4). The divergence time of Translucidithyrium is earlier than other genera in Phaeothecoidiellaceae. We estimate that the long divergence time should affect the genetic variation (Pauling 1964;Hall and Hallgrímsson 2008). Additionally, the evolutionary molecular clock approach confirmed the long clades of Translucidithyrium in the phylogenetic tree (Fig. 1).
Historical events amongst different biological groups could then be compared with the dates of plate tectonic movements and paleoecology, according to the corresponding geological time scale (Lomolino et al. 2006;Berbee and Taylor 2010). Through relevant studies on the Qinghai-Tibet Plateau, it was found that the time of intense tectonic uplift and denudation is concentrated in 60-35 Mya, 25-17 Mya, 12-8 Mya and 5 Mya. Global cooling might have an impact on climate change in East Asia, especially at 15 Mya and 8 Mya (Lu et al. 2010). Rising plateaus and global cooling were drying up Asia (Liu 2000;Garzione et al. 2015). The time of the Qinghai-Tibet Plateau uplift and global cooling corresponded to the interval of the species in Translucidithyrium divergence time. We predict that the speciation of T. chinense was earlier than the speciation of T. thailandicum, as the divergence of Translucidithyrium was related to the Qinghai-Tibet Plateau uplift and global cooling. According to the evolution history of Translucidithyrium, it could be speculated that the speciation of T. chinense was earlier than T. thailandicum. With the climate becoming colder and with increased drought, T. chinense migrated from China to Thailand gradually to find a suitable area, then T. thailandicum formed. Due to the end of global cooling, the distribution pattern of Translucidithyrium in two different countries formed. Increasing fresh collections and application of new methodologies may result in modified conclusions.